This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. After initial establishment of an endothelial network in vivo, the surrounding mesenchymal cells differentiate into mural cells, including smooth muscle cells (SMC) or pericytes, and are recruited to support the vascular network by stabilizing nascent endothelial vessels during mouse vascular development. However, it is unclear how human mesenchymal cells and SMC facilitate de novo human blood vessel formation and maturation. We recently found that mouse 10T1/2 cells (mouse embryonic fibroblast cell line with mesenchymal potential) enhanced human embryonic stem cell (hESC)-derived blood vessel formation in vivo. To understand the interactive relationship of endothelial cells (EC) and SMC during vascular development, we established an effective hESC system for the generation of CD34+ progenitor cells. Our preliminary studies show that CD34+ cells derived from hESCs have potential to become endothelial cells (EC) and SMCs. We propose the following Specific Aims: 1. To establish serum-free conditions and investigate factors that direct hESC generating EC and SMC from hESC. 2. To characterize vascular endothelial and SMC differentiation potential of CD34+ cells from hESCs. 3. To investigate the functions of human mural cells in supporting blood vessel development from hESCs. Our findings will have important implications for the possible future use of hESC in the tissue engineering of blood vessels for diseased human heart valves, and will provide a novel therapeutic option for ischemia or endothelial injury. Four NIH recent approved hESC lines, HUES 10, HUES 11, HUES 16, HUES 17 (NIHhESC-09-0023, 0024, 0029 and 0030) will be used.